Development of multivalent, ultrapotent nanobody cocktails for SARS-CoV-2 neutralization

项目总结/摘要 COVID-19的爆发严重影响了全球健康和经济。成本效益高， 迫切需要有效的治疗方法。骆驼科VHH抗体或纳米抗体（Nbs）是小的、高度特异性的， 稳定，易于生物工程化，并且可以快速和经济地从微生物制造。他们是 高度稳健并且对于给药是灵活的，包括可能通过雾化递送。综合这些 Nbs的独特性质使其可用于对抗呼吸道病毒，特别是SARS-CoV-2， 很吸引人。 我们最近开发了一种破坏性的蛋白质组学技术，用于大规模鉴定多表位，药物， 质量Nbs（Xiang et.等人，Cell Systems. 2021年）。使用这项技术，我们鉴定了> 8，000个高亲和力Nbs， SARS-CoV-2刺突（S）受体结合结构域（RBD），包括靶向高度中和 表位具有亚pM亲和力，可以中和浓度低于ng/ml的SARS-CoV-2， 前所未有的抗病毒抗体片段。结构蛋白质组学揭示了多个不同的表位， 潜在的中和机制。多表位和多价构建体的生物工程改善了 lng/ml（Xiang，et. al，科学。2020年）。最近，我们展示了 吸入治疗SARS的超有效和稳定的三聚体Nb构建体（PiN-21）的临床前功效- 在敏感的COVID-19模型中的CoV-2感染（Nambulli，et.《科学进展》2021年）。鼻内递送 0.6mg/kg的PiN-21显著降低了两个气道中的病毒负荷。关键是，PiN-21的气雾剂递送 在0.2 mg/kg时，肺病毒滴度降低6-log，最大限度地减少感染后肺病理学，并预防病毒感染。 肺炎结合显著的稳定性和低生产成本，这种创新疗法可能提供一种 方便和具有成本效益的选择，以减轻不断演变的流行病和未来的事件。 在修订版中，我们的目标是识别和表征对以下变体具有高度抗性的高效Nbs： 关注（VOCs）的SARS-CoV-2，研究中和机制的结构方法， 将超强效Nb构建体开发成安全有效的治疗剂。我们的中心假设是Nbs可以 被生物工程改造成多价和超有效的形式，以抵抗突变逃逸和 SARS-CoV-2的VOC。 完成我们建议的研究将导致成本效益和方便的COVID-19治疗 用于临床试验的候选人。高分辨率的结构研究将提供关键的见解， Nbs如何独特地靶向病毒以进行高亲和力结合和中和。关键是，该项目将作为 我们的多学科平台的测试平台，以开发未来有效的治疗和诊断试剂 由冠状病毒或其他病原体引起的流行病。

PROJECT SUMMARY/ABSTRACT The outbreak of COVID-19 has severely impacted global health and the economy. Cost-effective, highly efficacious therapeutics are urgently needed. Camelid VHH antibodies or nanobodies (Nbs) are small, highly stable, easily bioengineered, and can be rapidly and economically manufactured from microbes. They are highly robust and are flexible for administration, including possible delivery by nebulization. Together these unique properties of Nbs make their uses against respiratory viruses such as SARS-CoV-2 especially appealing. We recently developed a disruptive proteomic technology for large-scale identification of multi-epitope, drug- quality Nbs (Xiang et. al, Cell Systems. 2021). Using this technology, we identified > 8,000 high-affinity Nbs for the SARS-CoV-2 spike (S) receptor-binding domain (RBD) including Nbs that target highly neutralizing epitopes with sub-pM affinities and can neutralize SARS-CoV-2 at sub-ng/ml concentrations, which are unprecedented for antiviral antibody fragments. Structural proteomics revealed multiple distinct epitopes and potential neutralization mechanisms. Bioengineering of multi-epitope and multivalent constructs improved the potency to below 0.1 ng/ml (Xiang, et. al, Science. 2020). Most recently, we have demonstrated the high preclinical efficacy of an ultrapotent and stable trimeric Nb construct (PiN-21) for inhalation treatment of SARS- CoV-2 infection in a sensitive COVID-19 model (Nambulli, et. al, Science Advances. 2021). Intranasal delivery of PiN-21 at 0.6 mg/kg substantially reduces viral burdens in both airways. Critically, aerosol delivery of PiN-21 at 0.2 mg/kg decreases lung viral titers by 6-logs, minimizing lung pathology post-infection and preventing viral pneumonia. Combined with the marked stability and low production cost, this innovative therapy may provide a convenient and cost-effective option to mitigate the evolving pandemic and future events. In the revision, we aim to identify and characterize highly potent Nbs that are highly resistant to the variants of concern (VOCs) of SARS-CoV-2, investigate the neutralization mechanisms by structural approaches, and develop ultrapotent Nb constructs into safe and effective therapeutics. Our central hypothesis is that Nbs can be bioengineered into multivalent and ultrapotent forms to resist the mutational escape and the variants of concerns (VOCs) of SARS-CoV-2. Completion of our proposed studies will lead to cost-effective and convenient COVID-19 therapeutic candidates for translation into clinical trials. High-resolution structural studies will provide critical insights into how Nbs uniquely target the virus for high-affinity binding and neutralization. Critically, this project will serve as the testbed of our multidisciplinary platform to develop potent therapeutic and diagnostic reagents for future pandemics caused by coronaviruses or other pathogens.